Dario PasiniDipartimento di Chimica Organica

Università degli Studi di Pavia

APIB-2009Pavia, 3rd June 2009

Styrene-Based Copolymers as Soluble Platforms for the Biocatalytic Transformation of Organic

Substrates with Immobilized Enzymes

Overview

1) Biocatalysis, Solid Phase Synthesis and Soluble Polymers

2) Soluble Polymer-Achiral Substrate / Immobilized Enzyme

3) Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer

4) Conclusions and Outlook

General concepts involved in the use of supported organic targets and their biocatalytic

transformations

Crosslinked Polymers-Substrate / Free EnzymeConcept B

A. Basso, P. Braiuca, C. Ebert, L. Gardossi, P. Linda J. Chem. Technol. Biotechnol. 2006, 81, 1626-40

Tentagel and Argogel resins (polyethylene glycol chains grafted onto classical polystyrene/divinylbenzene cores)

•High swelling characteristics in aqueous solvents

•Low loading capacity

•Limited success in combination with biocatalysis

PEGA1900 (Copolymer Acrylamide/PEG) used in Enzymatic Solid Phase synthesis of peptides and resolution of racemates.

Biocatalysis and Solid Phase SynthesisConcept C versus Concept D

U. Grether, H. Waldmann Chem. Eur. J. 2001, 7, 959-971

N

MeO

O X

OO

O

Target Molecule

H

O

N

H

NH2

MeO

O X

OO

Target Molecule

O

N

H

NH

MeO

OO

O

N

HO

HX Target Molecule

O

HO

ImmobilizedPGA -

X = O, NH, NR

= Merrifield resin or Wang resins

+

Only when is a soluble linear polymer (PEG = polyethyleneglycol) high yields of the product could be achieved (Concept D)

Biocatalitically-TriggeredSafety-Catch Linker

Soluble Polymeric Supports

AdvantagesAdvantages

11- Easy monitoring of the support functional groups by - Easy monitoring of the support functional groups by common analytical techniques (e.g. common analytical techniques (e.g. 11H NMR)H NMR)22-Reactivity similar to the solution, homogeneous phase -Reactivity similar to the solution, homogeneous phase 3 3 – Facile product/reagent separation by precipitation of – Facile product/reagent separation by precipitation of the polymer in a non -solventthe polymer in a non -solvent

Soluble Polymeric Supports

D. E. Bergbreiter Chem. Rev. 2002, 102, 3345-3384

POLYSTYRENESPOLYSTYRENES

- Soluble in non polar organic solvents - Insoluble in MeOH- Good loading capacity

n

POLYETHYLENE GLYCOLSPOLYETHYLENE GLYCOLS

- Soluble in water and most organic solvents - Insoluble in diethyl ether- Low loading capacity

Soluble PS Copolymer-Substrate / Immobilized EnzymeConcept D

OH

ORO

O

R

O

x y

n i) Enzymatic hydrolysis (PGA)

n

O

OH

x y

Immobilized Enzyme

D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato Adv. Synth. Catal. 2007, 349, 971-978

+

iii) Recovery of the Copolymer by precipitation

iv) Isolation of substrate from the solution

ii) Filtration of the Enzyme

Soluble PS Copolymer -Substrate

Introduction of phenylacetic ester

monomers and copolymerization

with styrene at several loadings

O

O

On

+

n=1-3

O

O

On

x y

AIBN/70°C

Toluene

n=1-3x = 0.6-0.93

Monomer and Polymer Synthesis

Cl OOH

HO OH

NaOH/H2O

70°C

n=1-3

n O

O

On

OH

O

n

DICD/DPTS24 h

n=1-3

+

83-89%83-89% 70-90%70-90%

60-80%60-80%

xy

Characterization by 1H NMR Spectroscopy

O

Hb

HaHc

Hd

He

Hf

Hg

O

OHi

Hm HcHa Hb

Hd

Hi

Hm

He

Hf,g

1.52.02.53.03.54.04.55.05.56.06.57.07.58.0

O

80 20

O

O

Excellent agreement between feed and observed ratios of monomers

4 8 12 t (min)0

Solvent

PolymerAverage Molecular Mass

Mn

(number average)

Mw

(weight average)

PDIPolydispersity Index

Mw / MnValues between 1.5 and 2.4

Gel permeation chromatography

Properties as Supports

High Comonomer Loading (60:40)High Comonomer Loading (60:40) Bad precipitation in MeOH: Bad precipitation in MeOH: centrifugation neededcentrifugation needed

Low Comonomer Loading (93:7)Low Comonomer Loading (93:7) Excellent precipitation in MeOHExcellent precipitation in MeOH

Medium Comonomer Loading (80:20)Medium Comonomer Loading (80:20) Good precipitation in MeOHGood precipitation in MeOH

O

60 40

O

O

O

93 7

O

O

Sample Molecular Weight DistributionMn = 9080 ; Mw = 17630 ; PD = 1.9

O

80 20

O

O

Sample Molecular Weight DistributionMn = 11080 ; Mw = 18950 ; PD = 1.7

Copolymer Substrate Hydrolysis by PGA

Hydrolysis ConditionsHydrolysis Conditions-Temperature: 37°C-Temperature: 37°C-Mechanical stirring-Mechanical stirring-Mixed solvent system -Mixed solvent system (aqueous buffer 80/ (aqueous buffer 80/ DMF 20)DMF 20)

D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato, Adv. Synth. Catal., 2007, 349, 971– 978.

Co

nve

rsio

n(%

)

-0,4

-0,3

-0,2

-0,1

0

0 400 800 1200

ln((

P0-

P)/

P0)

0

10

20

30

0 400 800 1200t (s)

O

80 20

O

O

Immobilized on Eupergit (brown)Immobilized on Agarose (yellow)

Quantitative releaseFirst order kinetics

Enantiomeric Resolution Strategy

i)Enantioselective Enzymatic Cleavageii) Immobilized Enzyme Recoveryiii) Optically-Active Substrate and Soluble Copolymer Recovery

i) Chemical Cleavageii) Soluble Copolymer Recovery

Soluble Copolymer

(S,R)* (S,R)*

(S)*

Immobilized enzyme

Soluble Copolymer

(R)*

Soluble Copolymer

Possible application to enantioselective resolution of racemic carboxylic acids?

(R)*

(R)*Ch

emic

al R

efu

nct

ion

aliz

atio

n

Enzymatic Hydrolysis of (R,S)-Methyl mandelateConcept A

R = OMe, OEt, OPr,n, Opr,iso, OBut,n, NH2, NHPr,n, NHPr,iso

S. Rocchietti et al. Enzyme Microb. Technol. 2002, 31, 88-93

From E. Coli on activated agarose gel

Alternative Synthesis of Copolymer/Substrate

1 - Copolymerization

OOH

+

OOH

[ ]n [ ]m

O

[ ]n [ ]m

O

OOH

Good yields

Good purity

2 - Functionalization

HOOC OH

AIBN

toluene 70 °C48 h

1-DMAP / Pyridine / Me3SiCl 2- DMF / (COCl)2 a 0 °C

3- Et3N / CH2Cl2

m n

Efficient Polymer Functionalization: 1H NMR and IR

248 (ppm)6

Primary OH

Ester carbonyl

IR: KBr, diffuse reflectance,polymer powder

OO

OOH

[ ]85 [ ]15

AB

C

D

A+C BD

A B+C

OOH

[ ]85 [ ]15

AB

C

1H NMR: CDCl3, solution

Efficient Control of Polydispersity

OOH

[ ]85 [ ]15

OOH

AIBN

toluene 70 °C48 h

RAFT reagent

SSReversible Addition-FragmentationChain Transfer (RAFT) Polymerization

Functionalization“as usual”

O

[ ]85 [ ]15

O

OOH

+

Achieved control of Polydispersity:<1.2Achieved control of Degree of polymerization (50 to 500)

C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A 2008, 46, 5715-5723

Copolymer/Substrate Solubility Tests

Solvent Ratio (%) Solubility

MeCN 100 +

MeCN/H2O 50/50 -/+

DMF 100 +++

DMF/H2O 70/30 ++

DME 100 +++

DME/H2O 70/30 ++

DMSO 100 +

DMSO/H2O 50/50 -/+

Solvent Ratio (%) Solubility

MeCN 100 -

DMF 100 ++

DMA 100 +++

DMA/H20 80/20 ++

DMA/H20 20/80 +

DMSO 100 -/+

THF 100 +++

THF/H20 60/40 +

Phenylacetate Copolymer (R,S)-Mandelate Copolymer

DMF / Water Best Solvent

DMA / Water Best Solvent

Stability of Immobilized PGA in DMA/Water

0

20

40

60

80

100

120

0 500 1000 1500 2000

Res

idu

al a

ctiv

ity

%

Time (min)

20 % DMA

30 % DMA

60 % DMA

80 % DMA

Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer

Hydrolysis ConditionsHydrolysis Conditions

-Temperature: 25°C-Temperature: 25°C-Mechanical stirring-Mechanical stirring-Mixed solvent system -Mixed solvent system (aqueous buffer 80/ DMA 20)(aqueous buffer 80/ DMA 20)

Analytical Control

Conversion monitoringHPLC: Merck Hitachi LaChrom L-7000Column: AGILENT ZORBAX C18; 4,6 x 250mm = 220 nmFlow: 1 ml/minMethod (Gradient elution):A: 98% phosphate buffer 10 mM pH 3,2B: 2% CH3CNT = 25°C

Enantioselectivity monitoringHPLC: Merck Hitachi LaChrom L-7000Column: REGIS (S,S) Whelko-O1; 4,6 x 250mm = 220 nmFlow: 2 ml/minMethod:90% Hexane10 mM-10%Ammonium acetate 100 mM in EthanolT = 25°C

R

S

AcidsEsters

Preliminary Data Results

Immobilized PGA = 100U Immobilized PGA = 200U

Same Hydrolysis Conditions in Aqueous Buffer 80 / DMA 20

(R,S)-Mandelate - Copolymer

Hydrolysis Rate (mol/min) 0.04

Conversion (30h) 41%

ee% 18%

E 1.61

(R,S)-Methyl mandelate Free

Hydrolysis Rate (mol/min) 0.73

Conversion (5h) 43%

ee% 21%

E 1.77

Conclusions and Perspectives

1 – The use of Polystyrene Soluble Polymers as Tags for 1 – The use of Polystyrene Soluble Polymers as Tags for Substrates in combination with Immobilized Enzymes is Substrates in combination with Immobilized Enzymes is feasiblefeasible

2- In a biocatalytic reaction on a racemate, Enantioselectivity 2- In a biocatalytic reaction on a racemate, Enantioselectivity seems to be retained (more experiments needed to confirm seems to be retained (more experiments needed to confirm preliminary data)preliminary data)

3- Work-up, recovery and refunctionalization of the Soluble 3- Work-up, recovery and refunctionalization of the Soluble Polymer need to be optimizedPolymer need to be optimized

Acknowledgments

Dep. Organic Chemistry

Prof. Dario Pasini

Dr. Carmine Coluccini

Dr. Claudio Cornaggia

Michele Petenzi

Dep. Pharmaceutical Chemistry

Prof. Massimo Pregnolato

Prof. Daniela Ubiali

Dr. Teodora Bavaro

Dr. Davide A. Cecchini

Dr. Chiara Savarino

Visit: www.unipv.it/labt

Classical Synthesis of Copolymer/Substrate

1 –Functionalization of monomer

OO

OOH

AIBNtoluene 70 °C

48 h

- Difficult to precipitate

- Low yield

- Impurities

2 -Copolymerization

OOH

1-DMAP / Pyridine / Me3SiCl

CH2Cl2

2- DMF / (COCl)2 a 0 °C

HOOC OH ClOC OSiMe3DL-Mandelic Acid

Et3N / CH2Cl2

O

[ ]n [ ]m

O

OOH

80-85 %

+

n

m